1. Field of the Invention
This invention relates to the synthesis of proteins by the amplification, transcription and translation of a gene encoding the protein in an essentially cell-free medium.
2. Discussion of the Background
With the advent of the polymerase chain reaction (PCR), one is now able to produce numerous copies of DNA segments in a cell-free system, given an appropriate oligonucleotide sequence with which to start the PCR reaction. (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al, Science, vol. 230, p. 1350 (1985); Mullis et al, Methods Enzymo., vol. 155p. 335 (1987); Innis et al, "PCR Protocols: A Guide to Methods and Applications, " Academic Press New York, (1990); and Saiki et al, Science, vol. 239, p. 487 (1988)). Numerous modifications of the PCR reaction and applications to recombinant DNA technology have since been reported.
PCR has been applied to genomic amplification to enhance the sensitivity in sequencing analysis (GAWTS; Stoflet et al, Science, vol. 239, p. 491 (1988)) and to facilitate diagnosis Li et al, Nature, vol. 335, p. 414 (1988); and Brenner et al, Biotechniques, vol. 7, p. 1096 (1989)). PCR has also been utilized to facilitate site-directed mutagenesis (Kadowaki et al, Gene, vol. 76, p. 161 (1989); Ito et al, Gene, vol. 77, p. 51 (1989); and Hemsley et al, Nucleic Acids Res., vol. 17, p. 6545 (1989)) and recombinant gene preparation studies (Jones et al, Biotechniques, vol. 8, p. 178 (1900); and Horton et al, Gene, vol. 77, p. 61 (1989)).
In addition, PCR has been exploited to develop a quick subcloning method which has been used in the preparation of the appropriate DNA cassette to insert into an expression vector plasmid for overexpression of a protein in vivo (MacFerrin et al, Proc. Natl. Acad. Sci. U.S.A., vol. 87, p. 1937 (1990)).
Furthermore, generation of mutant protein without any in vivo manipulation has been achieved by incorporating a phage promoter and/or translation initiation signal into a PCR primer. In particular, the preparation of human factor IX fragments, with or without mutation, by a method in which the RNA was amplified by synthesizing the cDNA using reverse transcriptase followed by PCR amplification and then in vitro translation, was demonstrated as a method for sequencing nucleic acid segments from very small quantities of nucleic acids (RAWIT; Sarkar et al, Science, vol. 244, p. 331 (1989). No quantitative data for the amount of the translation product were reported.
The PCR reaction has also been applied to a procedure known as DNA amplification-restricted transcription-translation (DARTT; Mackow et al, Pro. Natl. Acad. Sci. USA, vol. 87, p. 518 (1990)), wherein PCR is used to amplify DNA segments encoding a series of amino-terminal and/or carboxy-terminal truncated polypeptides of the VP4 outer capsid protein from rhesus rotaviruses as a means for determining the binding sites of the VP4 capsid protein to monoclonal antibodies raised against a number of serologically distinct rotaviruses.
Catalytically active thymidine kinase was synthesized from a cloned vaccinia virus thymidine kinase gene amplified by PCR, then transcribed and translated in vitro, but no quantified data were provided for this procedure (Wilson et al, Gene, vol. 77, p. 69 (1989)). Further, a number of other polypeptide by-products were produced during this procedure. These by-products were noted but not discussed. The appearance of numerous byproducts resulting from in vitro translation certainly raises doubt with regard to any inherent predictability of success of in vitro translation alone, much less when used in conjunction with in vitro amplification and transcription.
Thus, there are no reports of the successful production of large quantities of a protein by a method in which PCR gene amplification is coupled with in vitro transcription and translation. In particular, there are no reports of the successful production of a large quantity of a catalytically active enzyme by such a method. Thus, there remains a need for a method of producing large quantities of a protein in a cell-free system.